Accumulating evidence demonstrates implication of Notch signaling in the maintenance of stem cells, while the underlying molecular mechanism has remained less clear. Previously, we demonstrated the critical role of Notch signaling in the maintenance of melanocyte stem cells. Conditional ablation of Notch signaling in the melanocyte lineage results in a severe coat color dilution, followed by intensive hair graying due to apoptotic elimination of melanocyte stem cells. Although hair graying represents the most common phenotype of human aging, it remains largely unknown how aging impacts on the maintenance of melanocyte stem cells. We have found that the hair pigmentation phenotype resulting from Notch inhibition was restored by the treatment with either an antioxidant agent or an inhibitor for the stress responsible p38 MAP kinase, suggesting the implication of oxidative stress in the elimination of melanocyte stem cells. We have also identified a novel target of Notch signaling, Cidea, which was originally identified as one of pro-apoptotic factors and has been recently implicated in mitochondria metabolism. As acceleration of mitochondria metabolism is the major cause of oxidative stress, we hypothesize that Notch signaling through repressing Cidea restricts mitochondria metabolism to protect melanocyte stem cells from oxidative stress. Therefore, the focus of this application is to obtain an experimental proof of the implication of Notch signaling acting through Cidea in the regulation of oxidative stress in melanocyte stem cells. To address this hypothesis, we propose following three specific aims: Aim 1. to delineate the role of Notch signaling in protecting of melanocyte stem cells against oxidative stress; Aim 2. to clarify the role of Cidea in the regulation oxidative stress in melanocyte stem cells; and Aim 3. to elucidate the physiological link between Notch signaling and Cidea in the regulation of oxidative stress in melanocyte stem cells using the combinatory gene knockout approach in mice. Our application will uncover a novel role of Notch signaling in the maintenance of stem cells, in which Notch signaling mediates stem cell resistance to oxidative stress to ensure long-term survival of stem cells. PUBLIC HEALTH RELEVANCE. Understanding of stem cell regulation is of great importance for their clinical applications. By employing melanocyte stem cells as a model system, we will clarify a novel molecular mechanism by which stem cells are protected from oxidative stress to be ensured long-term survival.